In the last fifteen years, the important roles of small non-coding RNAs in regulation in all organisms have been recognized and begun to be studied. Our laboratory, in collaboration with others, undertook two global searches for non-coding RNAs in E. coli, contributing significantly to the more than 80 regulatory RNAs that are now identified. A large number of these small RNAs (sRNAs) bind tightly to the RNA chaperone Hfq. We and others have shown that every RNA that binds tightly to Hfq acts by pairing with target mRNAs, regulating stability and translation of the mRNA, either positively or negatively. Our lab has studied a number of these sRNAs in detail. We have found that expression of each sRNA is regulated by different stress conditions, and that the sRNA plays an important role in adapting to stress. We have also examined the mechanism by which Hfq operates to allow sRNAs to act. The lab continues to investigate the in vivo roles of small RNAs. The sRNA RyhB is important for iron homeostasis, by down-regulating expression of non-essential iron binding proteins under iron limitation. Two other sRNA remodel the outer membrane under high osmolarity conditions, while another Hfq-binding RNA, is dependent on an alternative sigma factor, Sigma E, for transcription and down-regulates outer membrane proteins. These sRNAs are characteristic of many regulatory RNAs that regulate the cell surface, possibly important during infection. An additional outer membrane protein, OmpX, is down-regulated by CyaR, also an Hfq-binding RNA that is positively regulated by cyclic AMP and CRP. In addition to OmpX, believed to be important for cell adhesion, CyaR down-regulates the synthesis of other proteins, including LuxS, the synthase for a quorum-sensing molecule believed to work on a broad range of species. CyaR may help during escape from biofilms under poor nutrient conditions (low glucose). Consistent with the idea that all major regulatory systems may have small RNA components, another Hfq-binding RNA, named MgrR, is regulated by PhoP and PhoQ, a two-component system important for Salmonella virulence. PhoP and PhoQ activate synthesis of the RNA under low Magnesium conditions;the small RNA inactivates an enzyme for modification of the cell surface lipopolysaccharide, eptB, affecting the cells sensitivity to antimicrobial peptides such as polymyxin. This is the first example of regulation of an LPS modifying enzyme by sRNAs. Expression of the LPS modification enzyme is under complex additional regulation at the level of transcription;under some stress conditions, high levels of transcription overcome the sRNA-dependent silencing. This work as well as work in other labs underscores the variety of regulatory networks that sRNAs participate in. We have previously shown that two sRNAs, DsrA and RprA, positively regulate translation of the stress sigma factor RpoS. In other experiments, we have developed flexible and rapid methods for creating translational fusions to genes of interest, allowing us to use genetic screens to identify sRNA translational regulators. Because we believe that essentially all of the Hfq-dependent sRNAs have now been identified, a library of expression plasmids for each of these sRNAs has been made and can be used to rapidly screen for regulation of a given target fusion. Extending the work on translational regulation of RpoS, a third sRNA, RyhA, now renamed ArcZ, was found to strongly stimulate RpoS translation by direct pairing. ArcZ is regulated in response to switches from aerobic to anaerobic growth, by a well-known two-component regulatory system. In addition to three sRNAs that positively regulate RpoS by direct pairing, four others others negatively regulate RpoS. The most likely explanation for the negative regulation is competition for the Hfq RNA chaperone. Given that possibility, competition among sRNAs has now been studied in a variety of conditions. Induction of some, but not all, sRNAs can interfere with other regulators, suggesting a hierarchy of regulation;even the uninduced levels of some sRNAs modulate the effects of other sRNAs by reducing their ability to bind Hfq. Hfq binds both to sRNAs and to mRNAs, but the importance of mRNA binding was not well understood. Sarah Woodson and coworkers at Johns Hopkins University identified an Hfq binding site in the rpoS mRNA important for annealing of DsrA to rpoS in vitro. In collaboration with them, we showed that this site also plays a critical role in vivo, and that Hfqs major role is to stabilize the interaction of sRNA and mRNA. The same screening method has now identified multiple sRNAs that positively and negatively affect bacterial motility, by regulating a key transcriptional regulator. Motility is down-regulated when cells grow in biofilms and must be up-regulated to move out of biofilms. Multiple sRNA regulators of yet other important transcriptional regulators are also being identified, leading to a much more complete view of the network of bacterial regulatory signaling. Hfq is a hexamer of identical subunits, related in sequence and structure to Sm proteins in eukaryotes. While many mutations have been created in Hfq, these have generally been studied in vitro with purified mutant protein, and have not been compared for different sRNAs and their targets. In collaboration with G. Storz, NICHD, interesting hfq alleles have been studied with multiple sRNA:mRNA reporters in vivo;the results demonstrate that some mutants are defective only for some pairs, suggesting that there are multiple modes for Hfq to bind and act to stimulate pairing. In addition, the role of individual subunits in the hexamer had not been examined. We have created genes encoding covalently linked multimers of Hfq, allowing us to place mutations in given subunits. Initial studies suggest that some sites within Hfq need only be present on alternating subunits for full function, while others are needed on all subunits. In order to determine if factors other than Hfq are necessary for the action of these sRNAs, a genetic selection was developed to select for failure of two sRNAs to act. Among the mutations isolated were changes in conserved and essential amino acids in hfq and loss of function mutations in pnp, encoding polynucleotide phosphorylase. Polynucleotide phosphorylase (PNPase) is a 3 to 5 endonuclease that associates with the RNA degradosome, an RNAse known to be involved in degradation of sRNAs as well as their target mRNAs. pnp mutations lead to increased instability and decreased levels of multiple sRNAs, and this decreased accumulation may be sufficient to explain their failure to act. Our genetic analysis suggests that PNPase may play an unexpected role in protecting sRNAs from degradation, probably by regulating the activity of the RNA degradosome.In addition, in collaborative work with S. Leppla, NIAID, the Hfq proteins in B. anthracis are being characterized. Unique to B. anthracis and closely related organisms are three Hfq species;genetic analysis and microarrays show that two of these proteins are essential for sporulation of this pathogen, while the third Hfq negatively regulates sporulation. This system is providing new insight into Hfq function and a useful comparison to the E. coli work.